Genome-Wide Analysis of the GLK Gene Family and Its Expression at Different Leaf Ages in the Citrus Cultivar Kanpei.

The GLK gene family plays a crucial role in the regulation of chloroplast development and participates in chlorophyll synthesis. However, the precise mechanism by which GLK contributes to citrus's chlorophyll synthesis remains elusive. The GLK gene family causes variations in the photosynthetic capacity and chlorophyll synthesis of different citrus varieties. In this study, we identified tissue-specific members and the key CcGLKs involved in chlorophyll synthesis. A total of thirty CcGLK transcription factors (TFs) were discovered in the citrus genome, distributed across all nine chromosomes. The low occurrence of gene tandem duplication events and intronic variability suggests that intronic variation may be the primary mode of evolution for CcGLK TFs. Tissue-specific expression patterns were observed for various GLK family members; for instance, CcGLK12 and CcGLK15 were specifically expressed in the skin, while CcGLK30 was specific to the ovary, and CcGLK10, CcGLK6, CcGLK21, CcGLK2, CcGLK18, CcGLK9, CcGLK28, and CcGLK8 were specifically expressed in the leaves. CcGLK4, CcGLK5, CcGLK11, CcGLK23, CcGLKl7, CcGLK26, and CcGLK20 may participate in the regulation of the ALA, prochlorophylate, protoporphyrin IX, Mg-protoporphyrin IX, Chl b, T-Chl, MG-ProtoIX ME, and POR contents in citrus.

Effect of exogenous melatonin on antioxidant properties and fruit softening of 'Fengtang' plum fruit (Prunus salicina Lindl.) during storage at room temperature.

'Fengtang' plums soften quickly and lose flavor after harvest. This study comprehensively evaluated the effect of exogenous melatonin on the fruit quality of 'Fengtang' plums. According to our findings, exogenous melatonin prevented plum fruit from losing water, delayed the decline in firmness, and preserved a high TSS/TA level. Additionally, exogenous melatonin also enhanced the activity of antioxidant enzymes and increased the non-enzymatic antioxidants, thereby further increasing the antioxidant capacity of plum fruit. Notably, exogenous melatonin delayed the degradation of covalent soluble pectin (CSP), cellulose, and hemicellulose, as well as the rise in water-soluble pectin (WSP) concentration and the activity of cell wall degrading enzymes. Further investigation using atomic force microscopy (AFM) revealed that the chain-like structure of ionic-soluble pectin (ISP) and the self-assembly network structures of CSP were depolymerized, and melatonin treatment retarded the depolymerization of pectin structures. Our results showed that exogenous melatonin preserved the postharvest quality of plum fruits by controlling fruit softness and antioxidant capacity during storage.

Physiological Mechanisms of Citrus Fruit Cracking: Study on Cell Wall Components, Osmoregulatory Substances, and Antioxidant Enzyme Activities.

Fruit cracking affects both the yield and economic efficiency of citrus; however, the underlying mechanism remains unclear. Therefore, this study focused on resistant and susceptible cultivars to identify the mechanisms underlying fruit cracking. The results showed that in 'Mingrijian', pectin morphological transformation and hemicellulose and lignin degradation in the pericarp were important contributing factors. During the critical fruit-cracking period (115-150 days after flowering), the water-soluble pectin, protopectin, and lignin contents in the pericarp of 'Daya' presented inverse changes relative to those in 'Mingrijian', thus enhancing the mechanical properties and resistance of pericarp. From 115 to 150 days after flowering, the soluble sugar content in the pulp of 'Mingrijian' increased rapidly by 97.35%, aiding in pulp water absorption and expansion. Moreover, the soluble protein content in the pericarp of 'Mingrijian' exhibited a declining trend and was lower than that of 'Daya', thus affecting the overall metabolism. The superoxide dismutase (SOD) activity in the pericarp of 'Mingrijian' gradually decreased from 115 to 180 days after flowering, while the peroxidase (POD) activity remained at a low level, resulting in weaker antioxidant capacity and lower environmental resistance. This study provides valuable insights into the mechanisms of citrus fruit cracking, laying the foundation for preventive and control strategies.

Effect of Natural Variation and Rootstock on Fruit Quality and Volatile Organic Compounds of 'Kiyomi tangor' (Citrus reticulata Blanco) Citrus.

In this study, we compared the fruit quality and color of 'Kiyomi' (WT) and its mutant (MT) grafted on Ziyang xiangcheng (Cj) (WT/Cj, MT/Cj), and the MT grafted on Trifoliate orange (Pt) (MT/Pt). The differences in sugar, organic acid, flavonoids, phenols, and volatile substances of the three materials were also analyzed by high performance liquid chromatography (HPLC) and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results showed significant differences in the appearance of WT/Cj, MT/Cj, and MT/Pt. MT/Pt, compared to WT/Cj, MT/Cj, had lower sugar, acid, phenol and flavonoid contents in the pulp. However, MT/Pt pulp was higher in vitamin C (VC), and the peel had significantly higher total phenol and flavonoid contents. In terms of pulp, WT/Cj had the greatest diversity of volatile organic compounds (VOCs). 4-methyl-1-pentanol was significantly higher in MT/Cj pulp, while MT/Pt pulp had a unique octanoic acid, methyl ester. VOCs were more diverse in the peels of the three materials. ß-Myrcene and valencen were significantly higher in MT/Cj peels. In contrast, 16 unique VOCs were detected in MT/Pt, and D-limonene content was significantly higher than in WT/Cj and MT/Cj. The results suggest Trifoliate orange is a suitable rootstock for MT.

Comprehensive analyses of the citrus WRKY gene family involved in the metabolism of fruit sugars and organic acids.

Sugars and organic acids are the main factors determining the flavor of citrus fruit. The WRKY transcription factor family plays a vital role in plant growth and development. However, there are still few studies about the regulation of citrus WRKY transcription factors (CsWRKYs) on sugars and organic acids in citrus fruit. In this work, a genome-wide analysis of CsWRKYs was carried out in the citrus genome, and a total of 81 CsWRKYs were identified, which contained conserved WRKY motifs. Cis-regulatory element analysis revealed that most of the CsWRKY promoters contained several kinds of hormone-responsive and abiotic-responsive cis-elements. Furthermore, gene expression analysis and fruit quality determination showed that multiple CsWRKYs were closely linked to fruit sugars and organic acids with the development of citrus fruit. Notably, transcriptome co-expression network analysis further indicated that three CsWRKYs, namely, CsWRKY3, CsWRKY47, and CsWRKY46, co-expressed with multiple genes involved in various pathways, such as Pyruvate metabolism and Citrate cycle. These CsWRKYs may participate in the metabolism of fruit sugars and organic acids by regulating carbohydrate metabolism genes in citrus fruit. These findings provide comprehensive knowledge of the CsWRKY family on the regulation of fruit quality.

Effects of Exogenous Application of Glycine Betaine Treatment on 'Huangguoggan' Fruit during Postharvest Storage.

Loss of quality in citrus fruit is a common occurrence during postharvest storage due to oxidative stress and energy consumption. In recent years, glycine betaine (GB) has been widely applied to postharvest horticulture fruit. This study aimed to investigate the effect of GB treatment (10 mM and 20 mM) on the quality and antioxidant activity of 'Huangguogan' fruit during postharvest storage at room temperature. Our results indicated that both 10 mM and 20 mM treatments effectively reduced weight and firmness losses and maintained total soluble solid (TSS), titratable acidity (TA), and ascorbic acid contents. Additionally, GB treatment significantly increased the activity of antioxidant enzymes, maintained higher levels of total phenols and total flavonoids, and led to slower accumulation of H2O2. A transcriptome analysis conducted at 28 days after treatment (DAT)identified 391 differentially expressed genes (DEGs) between 20 mM GB (GB-2) and the control (CK) group. These DEGs were enriched in various pathways, particularly related to oxygen oxidoreductase, peroxidase activity, and flavonoid biosynthesis. Overall, the application of GB proved beneficial in enhancing the storability and extending the shelf life of 'Huangguogan' fruit.

Transcriptome and UPLC-MS/MS reveal mechanisms of amino acid biosynthesis in sweet orange 'Newhall' after different rootstocks grafting.

Sweet orange 'Newhall' (C. sinensis) is a popular fruit in high demand all over the world. Its peel and pulp are rich in a variety of nutrients and are widely used in catering, medicine, food and other industries. Grafting is commonly practiced in citrus production. Different rootstock types directly affect the fruit quality and nutritional flavor of citrus. However, the studies on citrus metabolites by grafting with different rootstocks are very limited, especially for amino acids (AAs). The preliminary test showed that there were significant differences in total amino acid content of two rootstocks (Poncirus trifoliata (CT) and C. junos Siebold ex Tanaka (CJ)) after grafting, and total amino acid content in the peel was higher than flesh. However, the molecular mechanism affecting amino acid differential accumulation remains unclear. Therefore, this study selected peel as the experimental material to reveal the amino acid components and differential accumulation mechanism of sweet orange 'Newhall' grafted with different rootstocks through combined transcriptome and metabolome analysis. Metabolome analysis identified 110 amino acids (AAs) and their derivatives in sweet orange 'Newhall' peels, with L-valine being the most abundant. L-asparagine was observed to be affected by both developmental periods and rootstock grafting. Weighted gene co-expression network analysis (WGCNA) combined with Redundancy Analysis (RDA) revealed eight hub structural genes and 41 transcription factors (TFs) that significantly influenced amino acid biosynthesis in sweet orange 'Newhall' peels. Our findings further highlight the significance of rootstock selection in enhancing the nutritional value of citrus fruits and might contribute to the development of functional citrus foods and nutritional amino acid supplements.

Acupuncture and moxibustion for endometriosis: A systematic review and analysis.

OBJECTIVES: This study aimed to examine the effect of acupuncture on symptoms and health-related quality of life in patients with endometriosis. METHODS: Nine biomedical databases were searched to April 2022 to identify randomized controlled trials of acupuncture and/or moxibustion used alone or as adjunct to guideline-recommended pharmacotherapy for the treatment of endometriosis. One reviewer extracted data and another verified the data. A random effects model was used to calculate mean differences. RESULTS: Fifteen trials involving 1018 patients met the inclusion criteria, but diversity in comparisons and outcome measures prevented meta-analysis. Compared to sham acupuncture, manual acupuncture was more effective at reducing dysmenorrhea VAS pain score (mean difference [MD] - 2.40, 95 % CI [- 2.80, - 2.00]; moderate certainty evidence), pelvic pain VAS score (MD - 2.65, 95 % CI [- 3.40, - 1.90]; high certainty evidence) and dyspareunia VAS scores (MD - 2.88, [- 3.83, - 1.93]), lessened the size of ovarian cyst (MD - 3.88, 95 % CI [- 7.06, - 0.70]), and improved quality of life. Compared to conventional therapy, manual acupuncture plus conventional therapy and warm needle alone resulted in greater improvements in quality of life than conventional therapy. Among the six studies that reported safety, fewer adverse events were reported in participants who received acupuncture or moxibustion. CONCLUSIONS: Low to moderate certainty evidence from single studies showed that manual acupuncture may improve pain-related symptoms and quality of life; however, there is insufficient evidence on the overall effectiveness of acupuncture and moxibustion for endometriosis.

Natural Variation Confers 'Aiyuan 38' Citrus Mutant a New Color and Unique Flavor.

Citrus exhibits unique nutritional values. Most citrus cultivars are derived from mutations. However, the effect of these mutations on fruit quality is unclear. We have previously found a yellowish bud mutant in the citrus cultivar 'Aiyuan 38'. Therefore, this study aimed to determine the effect of the mutation on fruit quality. 'Aiyuan 38' (WT) and a bud mutant variant (MT) were used to analyze variations in fruit color variation and flavor substances using colorimetric instruments, high-performance liquid chromatography (HPLC), headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS), and odor activity values (OAVs). The mutation in MT conferred yellowish characteristics to its peel. Although the differences in total sugar and acid content of the pulp were not statistically significant between WT and MT, the MT glucose content was significantly lower and the malic acid level was significantly higher. HS-SPME-GC-MS analysis revealed that the MT pulp released more types and contents of volatile organic compounds (VOCs) than the WT, whereas the opposite trend was observed for the peel. Analysis of the OAV revealed that the MT pulp contains 6 unique VOCs, whereas the peel contains only 1. This study provides a useful reference for the study of flavor substances associated with citrus bud mutations.

Comparative Transcriptomic and Physiological Analyses Reveal Key Factors for Interstocks to Improve Grafted Seedling Growth in Tangor.

Interstock is an important agronomic technique for regulating plant growth and fruit quality, and overcoming the incompatibility between rootstocks and scions; however, the underlying mechanisms remain largely unknown. In this study, the effects and regulatory mechanisms of tangor grafting, with and without interstocks, on the growth and development of scions were analyzed by combining morphology, physiology, anatomy and transcriptomics. Morphological and physiological analyses showed that interstocks ('Aiyuan 38' and 'Daya') significantly improved the growth of seedlings, effectively enhanced the foliar accumulation of chlorophyll and carotenoids, and increased the thickness of leaf tissues. Using 'Aiyuan 38' as the interstock, photosynthetic efficiency and starch content of citrus seedlings improved. Transcriptomics showed that genes related to photosynthesis and photosynthetic antenna proteins were upregulated in interstock-treated seedlings, with significant upregulation of photosystem PSI- and PSII-related genes. In addition, multiple key genes may be involved in plant hormone signaling, starch and sucrose metabolism, and transcriptional regulation. Taken together, these findings provide novel insights into the role of interstocks in regulating and contributing to the growth and development of grafted seedlings, and will further define and deploy candidate genes to explore the mechanisms of rootstock-interstock-scion interactions.

Analysis of Codon Usage Bias in Xyloglucan Endotransglycosylase (XET) Genes.

Xyloglucan endotransglycosylase (XET) genes are widely distributed in most plants, but the codon usage bias of XET genes has remained uncharacterized. Thus, we analyzed the codon usage bias using 4500 codons of 20 XET genes to elucidate the genetic and evolutionary patterns. Phylogenetic and hierarchical cluster analyses revealed that the 20 XET genes belonged to two groups. The closer the genetic distance, the more similar the codon usage preference. The codon usage bias of most XET genes was weak, but there was also some codon usage bias. AGA, AGG, AUC, and GUG were the top four codons (RSCU > 1.5) in the 20 XET genes. CitXET had a stronger codon usage bias, and there were eight optimal codons of CitXET (i.e., AGA, AUU, UCU, CUU, CCA, GCU, GUU, and AAA). The RSCU values underwent a correspondence analysis. The two main factors affecting codon usage bias (i.e., Axes 1 and 2) accounted for 54.8% and 17.6% of the total variation, respectively. Multiple correspondence analysis revealed that XET genes were widely distributed, with Group 1 genes being closer to Axis 1 than Group 2 genes, which were closer to Axis 2. Codons with A/U at the third codon position were distributed closer to Axis 1 than codons with G/C at the third codon position. PgXET, ZmXET, VlXET, VrXET, and PcXET were biased toward codons ending with G/C. In contrast, CitXET, DpXET, and BrpXET were strongly biased toward codons ending with A/U, indicating that these XET genes have a strong codon usage bias. Translational selection and base composition (especially A and U at the third codon position), followed by mutation pressure and natural selection, may be the most important factors affecting codon usage of 20 XET genes. These results may be useful in clarifying the codon usage bias of XET genes and the relevant evolutionary characteristics.